Conformational diseases are a group of disorders apparently unrelated to each other, but sharing a striking similarity in clinical presentations that reflect their shared molecular mechanisms of initiation and self-association, with consequent tissue deposition and damage.
The structural interest is due to the fact that these varied diseases each arise from an aberrant conformational transition in an underling protein, characteristically leading to protein aggregation and tissue deposition. Medically, the presentation of these conformational diseases reflects this molecular mechanism, with typically a slow and insidious onset when the transition is occurring in a normal protein, but a more sudden onset when it occurs in an unstable variant of the protein. Two examples of special significance of such conformational diseases are the Transmissible Spongiform Encephalopathies and Alzheimer dementia, a disease that threatens to overwhelm health care systems in the developed world (for a review see Carrell et al., 1997).
Transmissible spongiform encephalopathies (TSE) also known as prion diseases are a group of neurodegenerative diseases that affect humans and animals. Creutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straussler-Scheiker disease (GSS) and fatal familial insomnia (FFI) in humans as well as scrapie and bovine spongiform encephalopathy (BSE) in animals are some of the TSE diseases (Prusiner, 1991).
Although these diseases are relatively rare in humans, the risk for the transmissibility of BSE to humans through the chain food has taken the attention of the public health authorities and the scientific community (Cousens et al., 1997, Bruce et al., 1997).
These diseases are characterized by an extremely long incubation period, followed by a brief and invariably fatal clinical disease (Roos et al., 1973). To date no therapy is available.
The key characteristic of the disease is the formation of an abnormally shaped protein named PrPSc, which is a post-translationally modified version of a normal protein, termed PrPC (Cohen and Prusiner, 1998). Chemical differences have not been detected to distinguish between PrP isoforms (Stahl et al., 1993) and the conversion seems to involve a conformational change whereby the α-helical content of the normal protein diminishes and the amount of β-sheet increases (Pan et al., 1993). The structural changes are followed by alterations in the biochemical properties: PrPC is soluble in non-denaturing detergents, PrPSc is insoluble; PrPC is readily digested by proteases, while PrPSc is partially resistant, resulting in the formation of a N-terminally truncated fragment known as “PrPres” (Baldwin et al., 1995; Cohen and Prusiner, 1998), “PrP 27-30” (27-30 kDa) or “PK-resistant” (proteinase K resistant) form.
At present there is not an accurate diagnosis for TSE (WHO Report, 1998, Budka et al., 1995, Weber et al., 1997). Attempts to develop a diagnostic test for prion diseases are hampered by the apparent lack of an immune response to PrPSc. The clinical diagnosis of CJD is based upon the combination of subacute progressive dementia (less than 2 years), myoclonus, and multifocal neurological dysfunction, associated with a characteristic periodic electroencephalogram (EEG) (WHO Report, 1998, Weber et al., 1997). However, variant CJD (vCJD), most of the iatrogenic forms of CJD and up to 40% of the sporadic cases do not have the EEG abnormalities (Steinhoff et al., 1996). On average the accuracy of clinical diagnosis is around 60% for CJD and highly variable for other prion-related diseases. The clinical diagnosis is more accurate only at the late-stage of the disease when clear symptoms have developed (Weber et al., 1997).
Genetic analysis is useful for the diagnosis of inherited prion diseases, but these represent only 15% of the cases. Neuroimaging is useful only to exclude other conditions of rapidly progressive dementia due to structural lesions of the brain (Weber et al., 1997). The findings obtained by imaging of the brain by computed tomography (CT) and magnetic resonance imaging (MRI) depend mainly on the stage of the disease. CT is much less sensitive and in early phase no atrophy is detected in 80% of the cases (Galvez and Cartier, 1983). MRI hyperintense signals have been detected in the basal ganglia besides atrophy (Onofrji et al., 1993). Like the changes observed by CT, these alterations are by no means specific.
Recent data have identified several neuronal, astrocytic and glial proteins that are elevated in CJD (Jimi et al., 1992). The protein S-100, neuron specific isoenzyme and ubiquitin are significantly increased in the cerebrospinal fluid (CSF) in the early phase of disease with decreasing concentrations over the course of the illness (Jimi et al., 1992). A marker of neuronal death, the 14-3-3 protein, has been proposed as a specific and sensitive test for sporadic CJD (Hsich et al., 1996). However, it is not useful for the diagnosis of vCJD, and much less specific in the genetic forms. As the 14-3-3 protein may be present in the CSF of patients with other conditions, the test is not recommended by WHO as a general screening for CJD and is reserved to confirm the clinical diagnosis (WHO Report, 1998).
By combining clinical data with the biochemical markers a higher success in the diagnosis is achieved. However, according to the operational diagnosis currently in use in the European Surveillance of CJD, definitive diagnosis is established only by neuropathological examination and detection of PrPSc either by immunohistochemistry, histoblot or western blot (Weber et al., 1997, Budka et al., 1995).
Formation of PrPSc is not only the most likely cause of the disease, but it is also the best known marker. Detection of PrPSc in tissues and cells correlates widely with the disease and with the presence of TSE infectivity, and treatments that inactivate or eliminate TSE infectivity also eliminate PrPSc (Prusiner, 1991). The identification of PrPSc in human or animal tissues is considered key for TSE diagnosis (WHO Report, 1998). One important limitation to this approach is the sensitivity, since the amounts of PrPSc are high (enough for detection with conventional methods) only in the CNS at the late stages of the disease. However, it has been demonstrated that at earlier stages of the disease there is a generalized distribution of PrPSc (in low amounts), especially in the lymphoreticular system (Aguzzi, 1997). Indeed, the presence of PrPSc has been reported in palatine tonsillar tissue and appendix obtained from patients with vCJD (Hill et al., 1997). Although it is not known how early in the disease course tonsillar or appendix biopsy could be used in vCJD diagnosis, it has been shown that in sheep genetically susceptible to scrapie, PrPSc could be detected in tonsillar tissue presymptomatically and early in the incubation period. However, PrPSc has not been detected in these tissues so far in any cases of sporadic CJD or GSS (Kawashima et al., 1997).
The normal protein is expressed in white blood cells and platelets and therefore it is possible that some blood cells may contain PrPSc in affected individuals (Aguzzi, 1997). This raises the possibility of a blood test for CJD, but this would require an assay with a much greater degree of sensitivity than those currently available.
Prion replication is hypothesized to occur when PrPSc in the infecting inoculum interacts specifically with host PrPC, catalyzing its conversion to the pathogenic form of the protein (Cohen et al., 1994). This process takes from many months to years to reach a concentration of PrPSc enough to trigger the clinical symptoms.
The infective unit of PrPSc seems to be a β-sheet rich oligomeric structure, which converts the normal protein by integrating it into the growing aggregate (FIG. 1). The conversion has been mimicked in vitro by mixing purified PrPC with a 50-fold molar excess of previously denatured PrPSc (Kocisko et al., 1994).
The in vitro conversion systems described so far have low efficiency, since they require an excess of PrPSc and therefore are not useful for diagnostic purposes because they cannot monitor undetectable amounts of the marker. The reason for the low efficiency is that the number of PrPSc oligomers (converting units) remains fixed throughout the course of the assay. The converting units grow sequentially by the ends and as a result they become larger, but do not increase in number (FIG. 1).